Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-tocell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slowgrowing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.ultrahigh-throughput screening | microfluidic encapsulation | butyrylcholinesterase | Staphylococcus aureus | cell-cell interactions T he ultrahigh-throughput (1, 2) technique of screening (uHTS) in a double emulsion was applied in directed enzyme evolution (3, 4) to investigate the idea that a universal genotypephenotype linkage was provided by compartmentalization. Artificial compartments of double emulsions were produced with high polydispersity by shear stress (5, 6), which significantly decreased the portion of uniform droplets and thereby reduced the sensitivity and the maximal sorting rate. By contrast, sophisticated custom sorters demonstrated the screening of precise monodisperse droplets of water-in-oil emulsions generated by microfluidic technology (1, 7). However, it is not always convenient to use custom devices, and the use of oil as a continuous phase limits the sorting rate. Alternatively, compartmentalization in microfluidic double emulsion (MDE) enables uHTS of >10,000 events/s using commercially available cell sorters (8, 9). Furthermore, biocompatible oil and water phases provide viability and proliferation of Escherichia coli cells (10) inside the microenvironment of a double emulsion.Here, we propose an MDE-FACS platform that combines the benefits of previously reported systems based on compartmentalization in MDE and FACS selection together with modern omics (Fig. 1). We succeeded in assembling this platform using commercially available parts, which included straightforward microfluidics (Fig. S1) for MDE generation, multiparametric FACS for uHTS, next-generation sequencing (NGS) for bioinformatic predictions, and mass spectrometry for proteome and secretome analysis. We demonstrated this idea with several single-cell methods (Fig. S2), including the selection of di...

SignificanceAnalyzing complex microbial communities is the milestone of modern microbiology, calling for “deep functional profiling” techniques. While next generation sequencing revolutionized our understanding of microbiota communities, we still lack high-throughput technologies to precisely determine their functionality. Here we show how cultivation of individual bacteria inside droplets of microfluidic double water-in-oil-in-water emulsion enables us to isolate the clones with a desired activity. This approach allows us not only to select the potent antibiotic producer but also to discover a distinct mechanism of self-resistance as well as assess its efficiency on entire microbiomes. The outcome of this methodology shows that it could be effectively transferred to numerous applications in microbiology and biotechnology.

Liver metastasis is the main cause of colorectal cancer (CRC)-related death. Neutrophil extracellular traps (NETs) play important roles in CRC progression. Deoxyribonuclease I (DNase I) has been shown to alter NET function by cleaving DNA strands comprising the NET backbone. Moreover, DNase I displays high antimetastatic activity in multiple tumor models. To circumvent long-term daily administrations of recombinant DNase I, we have developed an adeno-associated virus (AAV) gene therapy vector to specifically express DNase I in the liver. In this study, we demonstrate AAV-mediated DNase I liver gene transfer following a single intravenous injection suppresses the development of liver metastases in a mouse model of CRC liver metastasis. Increased levels of neutrophils and NET formation in tumors are associated with poor prognosis in many patients with advanced cancers. Neutrophil infiltration and NET formation were inhibited in tumor tissues with AAV-DNase I treatment. This approach restored local immune responses at the tumor site by increasing the percentage of CD8 + T cells while keeping CD4 + T cells similar between AAV-DNase I and AAV-null treatments. Our data suggest that AAV-mediated DNase I liver gene transfer is a safe and effective modality to inhibit metastasis and represents a novel therapeutic strategy for CRC.

We report a novel family of natural lipoglycopeptides produced by Streptomyces sp. INA-Ac-5812. Two major components of the mixture, named gausemycins A and B, were isolated, and their structures were elucidated. The compounds are cyclic peptides with a unique peptide core and several remarkable structural features, including unusual positions of D-amino acids, lack of the Ca 2+ -binding Asp-X-Asp-Gly (DXDG) motif, tyrosine glycosylation with arabinose, presence of 2-amino-4-hydroxy-4-phenylbutyric acid (Ahpb) and chlorinated kynurenine (ClKyn), N-acylation of the ornithine side chain. These major components of the peptide antibiotic family have pronounced activity against Grampositive bacteria. The mechanism of action of gausemycins was explored by a number of methods, showing significant differences compared to glycopeptides and related lipopeptides. Gausemycins exhibit only slight Ca 2+ -dependence of antimicrobial activity and induce no pore formation at low concentrations. Moreover, there is no detectable accumulation of cell wall biosynthesis precursors under treatment with gausemycins.